Methods and compositions for treating cancer

ABSTRACT

This disclosure provides compositions and method useful for treating cell proliferative disorders including cancer. The disclosure provides cannabidiol derivatives and compositions thereof either alone or in combination with THC or a derivative thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/075,848, filed Nov. 8, 2013, which is a continuation of U.S.application Ser. No. 12/600,553, having a filing date of Apr. 26, 2010,which is a U.S. National Stage Application filed under 35 U.S.C. §371and claims priority to International Application No. PCT/US08/63837,filed May 16, 2008, which application claims priority to U.S.Provisional Application Ser. Nos. 60/938,635, filed May 17, 2007, and60/988,071, filed Nov. 14, 2007, the disclosures of which areincorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was funded by Grant Nos. CA102412 and CA82548 awarded byNational Institutes of Health. The government has certain rights in theinvention.

TECHNICAL FIELD

This invention relates to methods and compositions for treating cancer.More particularly, the invention provides cannabidiol derivatives andcompositions thereof.

BACKGROUND

The hemp plant Cannabis sativa, commonly referred to as marijuana, hasbeen used to alleviate symptoms of disease for thousands of years.Currently an oral formulation of Δ⁹-THC, the primary active cannabinoidconstituent of marijuana, is approved as an antiemetic agent for cancerpatients undergoing chemotherapy (Grinspoon, 1993). Additionally,studies suggest that cannabinoids may increase appetite and alleviatepain in the same patient population.

SUMMARY

The disclosure provides a method of treating cancer in a subject,comprising administering to a subject in need of such treatment atherapeutically effective amount of a composition comprising an agentthat modulates the expression and/or activity of an Id helix-loop-helixpolypeptide. In one embodiment, the agent is a cannabinoid, orderivative thereof including, for example, a cannabidiol or derivativethereof. In another embodiment, the cannabidiol or derivative thereofcomprises the structure of Formula I comprising an alkyl side chain andan open pyrane ring wherein the cannabidiol derivative inhibits Id-1expression, cancer cell proliferation, cancer cell invasion, metastasisor a combination thereof:

In yet another aspect, the R¹, R², R³, R⁴, are each independentlyselected from the group consisting of: H, —OH, aryl, substituted aryl,alkyl, substituted alkyl, carboxyl, aminocarbonyl,alkylsulfonylaminocarboxyl, and alkoxycarbonyl and wherein R is analkyl, or substituted alkyl of at least 6 carbon atoms. In oneembodiment, the cancer is an epithelial cell cancer such as, forexample, melanoma, breast carcinoma or lung carcinoma. In yet anotherembodiment, the cancer is brain cancer, such as glioblastoma multiforme.The method can further comprise administering a THC or derivativethereof. In another embodiment, the method can further includeadministering an effective amount of paclitaxel.

The disclosure also provides a method of treating cancer in a subject,comprising administering to a subject in need of such treatment atherapeutically effective amount of a composition comprising acannabidiol or derivative thereof and a THC or a derivative thereof. Inone embodiment, the cannabidiol or derivative thereof comprises thestructure of Formula I comprising an alkyl side chain and an open pyranering wherein the cannabidiol derivative inhibits Id-1 expression

In one aspect, R¹, R², R³, R⁴, are each independently selected from thegroup consisting of: H, —OH, aryl, substituted aryl, alkyl, substitutedalkyl, carboxyl, aminocarbonyl, alkylsulfonylaminocarboxyl, andalkoxycarbonyl and wherein R is an alkyl, or substituted alkyl of atleast 6 carbon atoms.

The disclosure also provides a method of treating cancer in a subject,comprising administering to a subject in need of such treatment atherapeutically effective amount of a composition comprising acombination of the cannabinoids cannabidiol (CBD) andtetrahydrocannabinol (THC).

The disclosure also provides a compound comprising the structure ofFormula I having an alkyl side chain and an open pyrane ring, wherein Rcomprises at least 6 carbon atoms

In one embodiment, R¹, R², R³, R⁴, are each independently selected fromthe group consisting of: H, —OH, aryl, substituted aryl, alkyl,substituted alkyl, carboxyl, aminocarbonyl, alkylsulfonylaminocarboxyl,and alkoxycarbonyl and wherein R is an alkyl, or substituted alkyl of atleast 6 carbon atoms.

The disclosure also provides a composition comprising a compound offormula I and a pharmaceutically acceptable carrier. The composition mayfurther comprise a tetrahydrocannabinol (THC) or a derivative thereof.

The disclosure also provides a composition comprising a combination ofthe cannabinoids cannabidiol (CBD) and tetrahydrocannabinol (THC). Inone embodiment, the composition further comprises a pharmaceuticallyacceptable carrier.

The disclosure demonstrates that the addition of CBD to Δ⁹-THC improvesthe overall potency and efficacy of Δ⁹-THC in the treatment of cancer(e.g., glioblasoma multiforme “GBM”).

In some aspects, Δ⁹-THC in combination with a lower concentration ofCBD, synergistically inhibits GBM cell growth and induces apoptosis. Theinhibitory properties of the combination were the result of activationof CB₂ receptors and a corresponding increase in oxygen radicalformation. The signal transduction mechanisms associated with theeffects of the combination treatment were significantly different fromthose observed with the individual compounds.

Accordingly, provided herein are methods and compositions for treatmentof cancer. Such compositions can comprise a combination of thecannabinoids cannabidiol (CBD) and tetrahydrocannabinol (THC).

One embodiment comprises a composition for treating cancer in a subject,the composition comprising a combination of the cannabinoids cannabidiol(CBD) and tetrahydrocannabinol (THC) or derivatives thereto.

In another embodiment, the composition can further include a compoundsuitable for treating a cell proliferation disorder, such as ananticancer drug (e.g. paclitaxel).

In another embodiment, the disclosure provides a method of treatingcancer in a subject, comprising administering to a subject in need ofsuch treatment a therapeutically effective amount of a compositioncomprising a combination of the cannabinoids cannabidiol (CBD) andtetrahydrocannabinol (THC).

The details of one or more embodiments of the disclosure are set forthin the accompanying drawings and the description below. Other features,objects, and advantages will be apparent from the description anddrawings, and from the claims.

BRIEF DESCRIPTION OF DRAWINGS

The accompanying drawings, which are incorporated into and constitute apart of this specification, illustrate one or more embodiments of theinvention and, together with the detailed description, serve to explainthe principles and implementations of the invention.

FIG. 1A-E provide data indicating that CBD is an effective inhibitor ofId-1 and corresponding breast cancer proliferation and invasiveness inMDA-MD231 cells. (A) depicts the results of a Boyden chamber invasionassay used to determine the effects of cannabinoids on the invasivenessof aggressive human breast cancer MDA-MB231 cells. Compounds were addedat concentrations of 0.1 μM, 1.0 μM, or 1.5 μM. Data are presented asrelative invasiveness of the cells through the Matrigel, where therespective controls are set as 100%. (B) depicts Western blot analysisof proteins from MDA-MB231 cells treated with vehicle (control), 0.1 μM,1.0 μM, or 1.5 μM of CBD for three days and analyzed as described below.(C) is a graph depicting the relative expression of Id-1 in treatedcells/vehicle cells. Proteins from MDA-MB231 cells treated withadditional cannabinoids for three days were extracted and analyzed forId-1 by Western blot analysis. Normalization was carried out bystripping the blots and re-probing with a monoclonal anti-tubulinantibody. (D) is a graph depicting the inhibitory effect of 1.5 μM CBDon Id-1 expression compared over a time course of one-, two-, andthree-days. (E) shows data depicting the structure activity relationshipof cannabinoids and the regulation of Id-1 protein expression. Proteinsfrom MDA-MB231 cells treated with vehicle (control) or 1.5 μM of variouscannabinoid compounds for two days and then analyzed for Id-1 by Westernblot analysis as described in the methods. A high molecular weightnon-specific band was used as a loading control (LC). Data are the meanof at least three replicates; bars, ±SE. Data were compared using aone-way ANOVA with a corresponding Dunnett's post-hoc test. (*)indicates statistically significant differences from control (p<0.05).

FIG. 2A-C provides data indicating that CBD inhibits the expression ofId-1 gene at the mRNA and promoter levels in MDA-MB231 cells. (A) showsthe inhibition of the Id-1 gene product (434 bp) by CBD. Expression ofthe β-actin gene product (232 bp) was used as a control. (B) showsluciferase activity in MDA-MB231 cells transiently transfected withId-1-sbsluc as determined in the presence of vehicle (control) or 1.5 μMCBD. Cells were treated for 2 days and luciferase activity was measured.(C) shows data for cells treated for 3 days. For both (B) and (C), allvalues were normalized for the amount of β-gal activity present in thecell extracts. Data are the mean of at least three replicates; bars,±SE. The data are represented as percentage of activity of the treatedcells/vehicle cells×100. Data were compared using the unpaired Student'st-test. (*) indicates statistically significant differences from control(p<0.05).

FIG. 3A-F provides data indicating that combinations of Δ⁹-THC and CBDproduce synergistic effects on the inhibition of cell growth in SF216and U251 cells but not in U87 cells. A 2×2 factorial design was used. A)depicts results for SF126, B) for U251, C) for U87MG cells that weretreated for three days with vehicle/no drug, Δ⁹-THC, CBD, or acombination of Δ⁹-THC and CBD. Concentrations of Δ⁹-THC and CBD thatproduce only minimal effects on cell proliferation (denoted low asopposed to high) were also tested in 2×2 factorial design in: D) forSF126, E) for U251 cells. Cell proliferation was measured using the MTTassay. The absorbance of the media alone at 570 nm was subtracted, and %control was calculated as the absorbance of the treated cells/controlcells×100. F) is a representative light microscope image of the effectsof the combination treatment on U251 cells from the experiment shown in(E) is presented (10×). Data are the mean of at least 3 independentexperiments; bars, ±SE.

FIG. 4A-C shows combination treatment of Δ⁹-THC and CBD specificallyinhibits ERK activity. The effects of cannabinoids on MAPK activity wereanalyzed using Western analysis. U251 cells were treated with vehicle ora combination of Δ9-THC (1.7 μM) and CBD (0.4 μM) for two days. Proteinswere extracted and analyzed for A) pERK and total ERK, B) pJNK 1/2 andp38 MAPK. U251 cells were treated with Δ9-THC (1.7 μM) and CBD (0.4 μM)alone and analyzed for C) pERK and total ERK. Either α-tubulin orβ-actin was used as a loading control (LC). Blots are representative ofat least 3 independent experiments.

FIG. 5 shows data indicating that when combined, Δ⁹-THC and CBD producegreater than additive effects on the cell cycle inhibition and inductionof apoptosis in U251 cells. U251 cells were treated for three days withΔ9-THC (1.7 μM), CBD (0.4 μM), or a 4:1 combination ratio [Δ9-THC (1.7μM)/CBD (0.4 μM)]. The number of cells staining positive for annexin(apoptosis) were measured using FACS analysis. % control was calculatedas positive annexin staining of the treated cells minus control cells.Data are the mean of at least 3 independent experiments; bars, ±SE. Datawere compared using a one-way ANOVA with a corresponding Tukey'spost-hoc test. (*) indicates statistically significant differences fromcontrol (p<0.05).

FIG. 6A-E shows CB₂ activation and corresponding increases in oxygenradical formation are involved in the inhibitory effects of thecannabinoid combination treatment. The number of U251 cells stainingpositive for annexin (apoptosis) after 3 days treatment were measuredusing FACS analysis. Cells were treated with A) a 4:1 combination ofΔ⁹-THC (1.7 μM) and CBD (0.4 μM) B) 2.5 μM Δ⁹-THC or C) 2.0 μM CBD inthe presence of 0.5 μM of the CB₁ antagonist, SR141716A (SR1), 0.5 μM ofthe CB₂ antagonist, SR144528 (SR2) or 20 μM α-tocopherol (TCP). %control was calculated as positive annexin staining of the treated cellsminus control cells. D) The effects of cannabinoids on pERK activitywere analyzed using Western analysis. α-tubulin was used as a loadingcontrol (LC). U251 cells were treated with vehicle or the indicateddrugs for three days. E) The production of cellular radical oxygenspecies (ROS)/H₂O₂ was measured using 2′7′Dichlorodihydrofluorescein andFACS analysis. U251 cells were treated with vehicle or a 4:1 combinationof Δ⁹-THC (1.7 μM) and CBD (0.4 μM). Data are the mean of at least 3independent experiments; bars, ±SE. Data were compared using a one-wayANOVA with a Bonferroni's multiple comparison post-hoc analyses. (*)indicates statistically significant differences from control (p<0.05).(#) indicates statistically significant differences from the combinationtreatment of THC/CBD (p<0.05).

FIG. 7A-C shows data indicating that ectopic expression of Id-1 blocksthe effect of CBD on MDA-MB231 invasiveness. (A) provides representativelight microscope images of control MDA-MB231 (−Id-1 cells, upper panels)and of MDA-MB231 cells that ectopically expressed Id-1 (+Id-1 cells,lower panels) after a two day treatment with vehicle (control) or 1.5 μMCBD, and then an overnight invasion assay. (B) provides data showing therelative invasiveness of the cells through the Matrigel, where therespective controls are set as 100%, and are the mean of at least threereplicates; bars, ±SE. Data were compared using the unpaired Student'st-test. (*) indicates statistically significant differences from control(p<0.05). (C) is a Western blot showing the inhibitory effect of CBD onId-1 expression in −Id-1 and +Id-1 MDA-MB231 cells was compared.

FIG. 8 shows that the combination treatment of Δ⁹-THC and CBD producesG1/S cell cycle arrest. Cell cycle was measured using PI staining andFACS analysis. U251 cells were treated for three days with Δ9-THC (1.7μM), CBD (0.4 μM), or a combination of Δ9-THC (1.7 μM) and CBD (0.4 μM).Cells were collected and analyzed using a desktop FACS Calibur with CellQuest Pro software. Modfit was used to determine the percentage of cellin G₀/G₁, S and G₂/G_(M) phase.

FIG. 9 shows that when combined, Δ⁹-THC and CBD produce a significantincrease in activation of multiple caspases. The effects of cannabinoidson caspase and p8 expression were analyzed using Western analysis. U251cells were treated for three days with Δ9-THC (1.7 μM), CBD (0.4 μM), ora combination of Δ9-THC (1.7 μM) and CBD (0.4 μM). Proteins wereextracted and analyzed for cleaved caspase 3, 7, 9 and PARP. Blots arerepresentative of at least 3 independent experiments.

FIG. 10 is a graph showing that CBD was also able to significantlyreduce the invasiveness of U251 cells.

FIG. 11 is a blot showing that treatment of U251 cells with CBD led to aconcentration-dependent inhibition of Id-1 protein expression.

DETAILED DESCRIPTION

As used herein and in the appended claims, the singular forms “a,”“and,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a compound”includes a plurality of such compounds and reference to “the cancer”includes reference to one or more cancers, and so forth.

Also, the use of “or” means “and/or” unless stated otherwise. Similarly,“comprise,” “comprises,” “comprising,” “include,” “includes,” and“including” are interchangeable and not intended to be limiting.

It is to be further understood that where descriptions of variousembodiments use the term “comprising,” those skilled in the art wouldunderstand that in some specific instances, an embodiment can bealternatively described using language “consisting essentially of” or“consisting of.”

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this disclosure belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice of the disclosed methods and compositions, the exemplarymethods, devices and materials are described herein.

Any publications discussed above and throughout the text are providedsolely for their disclosure prior to the filing date of the presentapplication. Nothing herein is to be construed as an admission that theinventors are not entitled to antedate such disclosure by virtue ofprior disclosure.

Activation of the two cannabinoid receptors, CB1 and CB2, can lead tothe inhibition of cell proliferation and induction of apoptosis inmultiple types of cancer cell lines resulting in the reduction of tumorgrowth in vivo (Guzman, 2003). The CB1 and CB2 receptors are members ofthe G-protein coupled receptor (GPCR) superfamily, and can interact withfive structurally distinct classes of compounds. These include theplant-derived classical cannabinoids, such as Δ9-THC and CBN; thenon-classical bicyclic cannabinoid agonists, such as CP55,940; theendogenous cannabinoid agonists, such as anandamide (AEA); and theaminoalkylindole (AAI) agonists, such as WIN55,212-2; and theantagonist/inverse agonists, such as SR141716A (Pertwee, 1997).

Interaction sites, independent of CB1 and CB2 receptors, may also beresponsible for the anticancer activity of cannabinoids (Ruiz et al.,1999; McAllister et al., 2005). There are more than 60 cannabinoids inCannabis sativa. In addition to Δ9-THC, the compounds cannabidiol (CBD),cannabinol (CBN), and cannabigerol (CBG) are also present in reasonablequantities (McPartland and Russo, 2001). CBN has low affinity for CB1and CB2 receptors, whereas the non-psychotropic cannabinoids, CBD andCBG, have negligible affinity for the cloned receptors.

Previous studies demonstrated that the helix-loop-helix protein Id-1, aninhibitor of basic helix-loop-helix (bHLH) transcription factors, playsa crucial role during breast cancer progression. Id-1 stimulatedproliferation, migration and invasion in breast cancer cells. Moreover,targeting Id-1 expression partially in breast cancer cells reducedinvasion and breast cancer metastasis in vitro and in preclinical animalmodels. The disclosure shows that Id-1 is a target for therapyapproaches, and that inhibiting Id-1 expression and/or activity providesa mechanism for treating patients with breast cancer. This approach maybe highly effective and safe in advanced breast cancer patients, given(1) the relationship between high Id-1 expression levels and aggressivebreast cancer cell behaviors; (2) partial reduction in Id-1 activity canachieve significant outcomes; and (3) Id-1 expression is low in normaladult tissues, thereby eliminating unwanted toxicities generallyassociated with currently available therapeutic modalities.

Id-1 protein plays a key role in the malignant progression of manyaggressive and invasive human cancers such as: leukemia, melanoma,hepatocellular carcinoma, colorectal adenocarcinoma, pancreatic cancer,lung cancer, kidney cancer, medullary thyroid cancer, papillary thyroidcancer, astrocytic tumor, neuroblastoma, Ewing's sarcoma, ovarian tumor,cervical cancer, endometrial carcinoma, breast cancer, prostate cancer,malignant seminoma, and squamous cell carcinomas, such as esophagealcancer, and head and neck cancer. Accordingly, Id-1 associated cellproliferative disorders include, but are not limited to, Leukemia,Melanoma, Squamous cell carcinoma (SCC) (e.g., head and neck,esophageal, and oral cavity), Hepatocellular carcinoma, Colorectaladenocarcinoma, Pancreatic cancer, Lung cancer, Kidney cancer, Medullarythyroid cancer, Papillary thyroid cancer, Astrocytic tumor,Neuroblastoma, Ewing's sarcoma, Ovarian tumor, Cervical cancer,Endometrial carcinoma, Breast cancer, Prostate cancer, and Malignantseminoma.

Approaches for targeting Id-1 expression include gene therapy usingantisense oligonucleotide, siRNA, non-viral or viral plasmid-basedstrategies. In addition, the development of new strategies to modulateId-1 expression/functional activity include the identification of smallmolecules that modulate the activity of Id-1. A range of small moleculesthat target the molecular pathology of cancer are now being developed,and a significant number of them are being tested in ongoing humanclinical trials. The disclosure demonstrates that cannabidiol (CBD) andCBD derivatives are inhibitors of Id-1. The use of CBD, and derivativesthereof, represents a novel strategy for the treatment of cancer.

As used herein, the term “CBD” and “CBD derivatives” includescannabinoids and derivatives thereof such as cannabidiol. Cannabinoidsare a group of terpenophenolic compounds present in Cannabis sativa. Theterm “cannabinoids” generally refers to a group of substances that arestructurally related to tetrahydrocannabinol (THC) or that bind tocannabinoid receptors. Plant cannabinoids are stable compounds with lowtoxicity profiles that are well tolerated by animals and humans duringchronic administration. A variety of chemical classes of cannabinoidsare useful in the methods provided herein including cannabinoidsstructurally related to THC, aminoalkylindoles, the eicosanoids relatedto the endocannabinoids, 1,5-diarylpyrazoles, quinolines andarylsulphonamides and additional compounds that do not fall into thesestandard classes but bind to cannabinoid receptors. Exemplary structuresare set forth below.

Data provided herein indicates that CBD and derivatives thereof that actas Id-1 inhibitor effectively inhibit genotypic and phenotypic changesthat allow aggressive breast cancers to proliferate, invade andmetastasize.

Since CBD inhibits Id-1 expression in aggressive breast cancer, thedisclosure also provides a rational drug design strategy and compoundsobtained there from as potent and efficacious analogs. The disclosuredemonstrates that the opened tetrahydropyran ring in CBD and aliphaticside chain of CBD are key pharmacophores involved in the inhibition ofId-1, alterations of these functional groups allow one to improve boththe potency and efficacy of the parent compound, CBD.

Moreover, reducing Id-1 expression with cannabinoids provides atherapeutic strategy for the treatment of additional aggressive cancerssince Id-1 expression was found to be up-regulated during theprogression of almost all types of solid tumors investigated.

Accordingly, provided herein are methods for modulating the activity ofa metastatic cell by regulating the activity of a target Id-1 using aCBD or CBD derivative. Methods can also include “regulating the activityof a target Id-1” includes: 1) mechanisms for modulating endogenousnucleic acid sequences that encode a target Id-1 such that Id-1polypeptide levels are decreased in a cell; 2) introducing exogenousnucleic acid sequences that inhibit Id-1 expression in a cell; 3)increasing the turnover rate of endogenous Id-1 polypeptides such thatId-1 polypeptide levels are decreased in a cell.

In some embodiments the methods described herein can be designed toidentify substances that modulate the biological activity of a Id-1 bymodulating the expression of an Id-1 nucleic acid sequence encoding anId-1 polypeptide. For example, methods can be utilized to identifycompounds that bind to Id-1 regulatory sequences. Alternatively, methodscan be designed to identify substances that modulate the biologicalactivity of a Id-1 by affecting the half-life of a Id-1 polypeptide.

In other embodiments, methods for treating a cell proliferation-relateddisorder are provided herein. Agents, substances or compounds thatregulate the expression and/or activity of endogenous Id-1 or thehalf-life of endogenous Id-1 may be used for treating a cellproliferation-related disorder. In general these methods can be used inthe treatment of conditions associated with disorders related toneoplastic cells and the metastasis thereof.

For administration to a subject, modulators of Id helix-loop-helixexpression and/or activity (e.g., inhibitory agents, nucleic acidmolecules, proteins, or compounds identified as modulators of a Idexpression and/or activity) will preferably be incorporated intopharmaceutical compositions suitable for administration.

The disclosure also pertains to the field of predictive medicine inwhich diagnostic assays, prognostic assays, pharmacogenomics, andmonitoring clinical trails are used for prognostic (predictive) purposesto thereby treat an individual prophylactically. As such, the disclosurecontemplates use of methods provided herein to screen, diagnose, stage,prevent and/or treat disorders characterized by expression orover-expression of an Id helix-loop-helix polypeptide, such as Id-1.Accordingly, a subject can be screened to determine the level of aparticular Id's expression or activity. A subject can also be screenedfor the susceptibility of neoplastic cells to techniques that inhibitthe expression or over expression of an Id polypeptide.

The disclosure also provides for prognostic (or predictive) assays fordetermining whether an individual is at risk of developing a disorderassociated with under expression of a target Id polypeptide. Such assayscan be used for prognostic or predictive purpose to therebyprophylactically treat an individual prior to the onset of a disordercharacterized by or associated with over expression or activity of atarget Id polypeptide, such as Id-1.

The disclosure provides cannabinoid derivatives (e.g., cannabidiols). Inone embodiment, the disclosure provides compositions comprisingcannabinoids cannabidiol (CBD) either alone or in combination withtetrahydrocannabinol (THC) or a derivative thereof. As described hereinCBD and derivatives thereof are significantly more effective than othercannabinoid compounds at inhibiting the expression of genes and proteinsthat modulate cancer aggressiveness (e.g. Id-1) in, for example, breastcancer aggressiveness. The data also indicated that Id-1 is a key factorwhose expression needed to be down-regulated in order for CBD to inhibitbreast cancer cell aggressiveness. The down-regulation of geneexpression produced by CBD was the result of the inhibition of theendogenous Id-1 promoter and its corresponding mRNA and protein levels.As shown in FIG. 1, CBD and derivatives thereof effectively inhibits theexpression of Id-1 in MDA-MB231 metastatic breast cancer cells.

To determine general structural components of CBD that were responsiblefor its inhibitory activity, CBD was compared against structurallyrelated cannabinoid compounds for their ability to inhibit Id-1 (FIG.1). THC had no activity against Id-1. Δ9THC (THC) is structurallyrelated to CBD with the primary exception being that the B ring or1,1′-di-methyl-pyrane ring (FIG. 1E) of THC has been opened in CBD.CP55,940 (Formula IV, below) was a compound that inhibited Id-1expression, however, it was still less effective than CBD. CP55,940 doeshave a open pyrane ring but the constituents within this area aresignificantly different compared to those present in CBD. Additionally,the alkyl chain is six carbons in length and contains a dimethyl heptyladdition. The data provided herein show that the unique activity (Id-1inhibition) is related to the opened pyrane ring and the possession ofan extended alkyl side chain.

The disclosure provides cannabidiol derivatives comprising an alkyl sidechain and an open pyrane ring wherein the cannabidiol derivativeinhibits Id-1. A general CBD derivative of the disclosure is set forthin Formula I, below:

wherein R, R¹, R², R³, R⁴, are each independently selected from thegroup consisting of: H, aryl, substituted aryl, alkyl, substitutedalkyl, carboxyl, aminocarbonyl, alkylsulfonylaminocarboxyl, andalkoxycarbonyl.

Alkyl groups include straight-chain, branched and cyclic alkyl groups.Alkyl groups include those having from 1 to 20 carbon atoms. Alkylgroups include small alkyl groups having 1 to 3 carbon atoms. Alkylgroups include medium length alkyl groups having from 4-10 carbon atoms.Alkyl groups include long alkyl groups having more than 10 carbon atoms.Cyclic alkyl groups include those having one or more rings. Cyclic alkylgroups include those having a 3-, 4-, 5-, 6-, 7-, 8-, 9- or 10-membercarbon ring. The carbon rings in cyclic alkyl groups can also carryalkyl groups. Cyclic alkyl groups can include bicyclic and tricyclicalkyl groups. Alkyl groups optionally include substituted alkyl groups.Substituted alkyl groups include among others those which aresubstituted with aryl groups, which in turn can be optionallysubstituted. Specific alkyl groups include methyl, ethyl, n-propyl,iso-propyl, cyclopropyl, n-butyl, s-butyl, t-butyl, cyclobutyl,n-pentyl, branched-pentyl, cyclopentyl, n-hexyl, branched hexyl, andcyclohexyl groups, all of which are optionally substituted.

Alkenyl groups include straight-chain, branched and cyclic alkenylgroups. Alkenyl groups include those having 1, 2 or more double bondsand those in which two or more of the double bonds are conjugated doublebonds. Alkenyl groups include those having from 2 to 20 carbon atoms.Alkenyl groups include small alkyl groups having 2 to 3 carbon atoms.Alkenyl groups include medium length alkenyl groups having from 4-10carbon atoms. Alkenyl groups include long alkenyl groups having morethan 10 carbon atoms, particularly those having 10-20 carbon atoms.Cyclic alkenyl groups include those having one or more rings. Cyclicalkenyl groups include those in which a double bond is in the ring or inan alkenyl group attached to a ring. Cyclic alkenyl groups include thosehaving a 3-, 4-, 5-, 6-, 7-, 8-, 9- or 10-member carbon ring. The carbonrings in cyclic alkenyl groups can also carry alkyl groups. Cyclicalkenyl groups can include bicyclic and tricyclic alkyl groups. Alkenylgroups are optionally substituted. Substituted alkenyl groups includeamong others those which are substituted with alkyl or aryl groups,which groups in turn can be optionally substituted.

Aryl groups include groups having one or more 5- or 6-member aromatic orheteroaromatic rings. Aryl groups can contain one or more fused aromaticrings. Heteroaromatic rings can include one or more N, O, or S atoms inthe ring. Heteroaromatic rings can include those with one, two or threeN, those with one or two O, and those with one or two S. Aryl groups areoptionally substituted. Substituted aryl groups include among othersthose which are substituted with alkyl or alkenyl groups, which groupsin turn can be optionally substituted.

Arylalkyl groups are alkyl groups substituted with one or more arylgroups wherein the alkyl groups optionally carry additional substituentsand the aryl groups are optionally substituted. Specific alkylarylgroups are phenyl-substituted alkyl groups, e.g., phenylmethyl groups.

Alkylaryl groups are aryl groups substituted with one or more alkylgroups wherein the alkyl groups optionally carry additional substituentsand the aryl groups are optionally substituted.

Optional substitution of any alkyl, alkenyl and aryl groups includessubstitution with one or more of the following substituents: halogens,—CN, —COOR, —OR, —COR, —OCOOR, —CON(R)₂, —OCON(R)₂, —N(R)₂, —NO₂, —SR,—SO₂R, —SO₂N(R)₂ or —SOR groups. Optional substitution of alkyl groupsincludes substitution with one or more alkenyl groups, aryl groups orboth, wherein the alkenyl groups or aryl groups are optionallysubstituted. Optional substitution of alkenyl groups includessubstitution with one or more alkyl groups, aryl groups, or both,wherein the alkyl groups or aryl groups are optionally substituted.Optional substitution of aryl groups includes substitution of the arylring with one or more alkyl groups, alkenyl groups, or both, wherein thealkyl groups or alkenyl groups are optionally substituted.

Optional substituents for alkyl, alkenyl and aryl groups include amongothers:

—COOR where R is a hydrogen or an alkyl group or an aryl group and morespecifically where R is methyl, ethyl, propyl, butyl, or phenyl groupsall of which are optionally substituted;

—COR where R is a hydrogen, or an alkyl group or an aryl groups and morespecifically where R is methyl, ethyl, propyl, butyl, or phenyl groupsall of which groups are optionally substituted;

—CON(R)₂ where each R, independently of each other R, is a hydrogen oran alkyl group or an aryl group and more specifically where R is methyl,ethyl, propyl, butyl, or phenyl groups all of which groups areoptionally substituted; R and R can form a ring which may contain one ormore double bonds;

—OCON(R)₂ where each R, independently of each other R, is a hydrogen oran alkyl group or an aryl group and more specifically where R is methyl,ethyl, propyl, butyl, or phenyl groups all of which groups areoptionally substituted; R and R can form a ring which may contain one ormore double bonds;

—N(R)₂ where each R, independently of each other R, is a hydrogen, or analkyl group, acyl group or an aryl group and more specifically where Ris methyl, ethyl, propyl, butyl, or phenyl or acetyl groups all of whichare optionally substituted; or R and R can form a ring which may containone or more double bonds;

—SR, —SO₂R, or —SOR where R is an alkyl group or an aryl groups and morespecifically where R is methyl, ethyl, propyl, butyl, phenyl groups allof which are optionally substituted; for —SR, R can be hydrogen;

—OCOOR where R is an alkyl group or an aryl groups;

—SO₂N(R)₂ where R is a hydrogen, an alkyl group, or an aryl group and Rand R can form a ring;

—OR where R═H, alkyl, aryl, or acyl; for example, R can be an acylyielding —OCOR* where R* is a hydrogen or an alkyl group or an arylgroup and more specifically where R* is methyl, ethyl, propyl, butyl, orphenyl groups all of which groups are optionally substituted.

Specific substituted alkyl groups include haloalkyl groups, particularlytrihalomethyl groups and specifically trifluoromethyl groups. Specificsubstituted aryl groups include mono-, di-, tri, tetra- andpentahalo-substituted phenyl groups; mono-, di-, tri-, tetra-, penta-,hexa-, and hepta-halo-substituted naphthalene groups; 3- or4-halo-substituted phenyl groups, 3- or 4-alkyl-substituted phenylgroups, 3- or 4-alkoxy-substituted phenyl groups, 3- or4-RCO-substituted phenyl, 5- or 6-halo-substituted naphthalene groups.More specifically, substituted aryl groups include acetylphenyl groups,particularly 4-acetylphenyl groups; fluorophenyl groups, particularly3-fluorophenyl and 4-fluorophenyl groups; chlorophenyl groups,particularly 3-chlorophenyl and 4-chlorophenyl groups; methylphenylgroups, particularly 4-methylphenyl groups, and methoxyphenyl groups,particularly 4-methoxyphenyl groups.

Pharmaceutically acceptable salts of CBD and CBD derivatives of thedisclosure are also included. For example, a pharmaceutically acceptablesalt can comprise pharmaceutically-acceptable anions and/or cations.Pharmaceutically-acceptable cations include among others, alkali metalcations (e.g., Li⁺, Na⁺, K⁺), alkaline earth metal cations (e.g., Ca²⁺,Mg²⁺), non-toxic heavy metal cations and ammonium (NH⁴⁺) and substitutedammonium (N(R′)₄ ⁺, where R′ is hydrogen, alkyl, or substituted alkyl,i.e., including, methyl, ethyl, or hydroxyethyl, specifically, trimethylammonium, triethyl ammonium, and triethanol ammonium cations).Pharmaceutically-acceptable anions include among other halides (e.g.,Cl⁻, Br⁻), sulfate, acetates (e.g., acetate, trifluoroacetate),ascorbates, aspartates, benzoates, citrates, and lactate.

Compounds of the disclosure can have prodrug forms. Prodrugs of thecompounds are useful in the methods of this disclosure. Any compoundthat will be converted in vivo to provide a biologically,pharmaceutically or therapeutically active form of a compound of thedisclosure is a prodrug. Various examples and forms of prodrugs are wellknown in the art. Examples of prodrugs are found, inter alia, in Designof Prodrugs, edited by H. Bundgaard, (Elsevier, 1985), Methods inEnzymology, Vol. 42, at pp. 309-396, edited by K. Widder, et. al.(Academic Press, 1985); A Textbook of Drug Design and Development,edited by Krosgaard-Larsen and H. Bundgaard, Chapter 5, “Design andApplication of Prodrugs,” by H. Bundgaard, at pp. 113-191, 1991); H.Bundgaard, Advanced Drug Delivery Reviews, Vol. 8, p. 1-38 (1992); H.Bundgaard, et al., Journal of Pharmaceutical Sciences, Vol. 77, p. 285(1988); and Nogrady (1985) Medicinal Chemistry A Biochemical Approach,Oxford University Press, New York, pages 388-392).

In one embodiment, the CBD or CBD derivative comprises Formula I,wherein R is an alkyl comprising from 2-10 carbon atoms. Typically thealkyl will comprise at least 6 carbon atoms (e.g., 6, 7, 8, 9 or morecarbon atoms). In some embodiments, the compound comprises an R₁, R₂,R₃, or R₄ that is the same as CBD (e.g., R₁ comprises a branched alkene,R₃ comprises a methyl group, R₂ and R₄ are hydroxyl groups).Alternatively, R₁ can be a branched chain alkyl and R₂ can be a methylgroup. In yet another embodiment, the compound of Formula I comprises anR group selected from a group consisting of:

In yet another embodiment, R is selected from a member of the groupabove and R₃ is an ethyl group.

In another embodiment, the disclosure provides a compound of Formula Iwherein R comprises a dimethylheptyl (DMH) analog or derivative thereof.In yet a further embodiment, the disclosure provides a compound ofFormula I, wherein R comprises a DMH analog or derivative thereof and R₃comprises an ethyl group.

Exemplary CBD compounds and CBD derivative compounds are set forth belowand it will be recognized that such compounds can include prodrugs,salts and ethers thereof.

Type Skeleton Cyclization Cannabigerol-type CBG

Cannabichromene-type CBC

Cannabidiol-type CBD

Tetrahydrocannabinol- and Cannabinol-type THC, CBN

Cannabielsoin-type CBE

iso- Tetrahydrocannabinol- type iso-THC

Cannabicyclol-type CBL

Cannabicitran-type CBT

Cannabigerol-type (CBG)

Cannabichromene-type (CBC)

Cannabidiol-type (CBD)

Cannabinodiol-type (CBND)

Tetrahydrocannabinol-type (THC)

Cannabinol-type (CBN)

Cannabitriol-type (CBT)

Cannabielsoin-type (CBE)

Isocannabinoids

Cannabicyclol-type (CBL)

Cannabicitran-type (CBT)

Cannabichromanone-type (CBCN)

Miscellaneous

The disclosure demonstrates the antiproliferative activities of threegroups of cannabinoid compounds. The groups included: 1) naturalcannabis constituents that have affinity for CB₁ and CB₂ receptors,Δ⁹-THC and CBN; 2) synthetic cannabinoid analogs that have high affinityfor CB₁ and CB₂ receptors, WIN 55,212-2 and CP55,940; and 3) naturalcannabis constituents that do not have appreciable affinity for CB₁ andCB₂ receptors, CBD and CBG. Breast cancer cells were treated for threedays and IC₅₀ values were calculated and provided in Table 1 below:

Compound MDA-MB231 MDA-MB436 Δ⁹-THC 1.2 (1.0-1.4) 2.5 (1.8-3.4) CBN 1.2(0.9-1.5) 2.6 (1.8-3.7) WIN 55, 212-2 1.7 (1.5-2.2) 2.4 (1.6-3.4) CP 55,940 2.5 (1.5-4.1) 1.3 (0.7-1.6) CBD 1.3 (1.0-1.9) 1.5 (1.0-1.9) CBG 2.3(2.1-2.5) 2.1 (1.5-3.0)

The rank order of potencies for the anti-proliferative effects of thecannabinoids in MDA-MB231 cells was:CBD=Δ⁹-THC=CBN>WIN55,212-2>CBG=CP55,940. The rank order of potencies forthe antiproliferative effects of the cannabinoids in MDA-MB436 cellswas: CBD=CP55,940>CBG=WIN55,212-2=Δ9-THC=CBN. The data demonstrates thatcannabidiol (CBD) is an effective inhibitor of human breast cancer cellaggressiveness, invasiveness, and therefore metastasis.

Invasion is an important step towards breast cancer cell metastasis.Therefore, the effects of several cannabinoids were tested on theirability to modulate the migratory and invasiveness activity of the mostaggressive human breast cancer cell line, MDA-MB231, in a reconstitutedbasement membrane in a Boyden chamber. All three compounds tested, i.e.,CBD, Δ⁹-THC, and WIN 55,212-2, significantly reduced the invasion ofMDA-MB231 cells (FIG. 1, panel A). Again, as was observed with the cellaggressiveness and invasiveness experiments, the most potent inhibitorof invasion was CBD. The IC₅₀ value and corresponding confidence limitsfor CBD were 260 nM (110-610).

The compounds described herein and compositions comprising the compoundsare useful to modulate the expression and/or activity of Id-1 inproliferating cells. In one exemplary embodiment, the informationprovided herein demonstrates a role for cannabidiols, and derivativesthereof, in inhibiting the metastasis through inhibition of Id-1expression and/or activity.

Accordingly, the compositions and CBD compounds can be used in methodsfor modulating metastatic cancer cell progression by regulating theexpression and/or activity of Id-1. The methods include using apharmaceutical composition that includes an agent that modulates theexpression and/or activity of Id-1. Exemplary agents include cannabinoidderivatives, such as cannabidiol and derivatives thereof.

U.S. patent application Ser. No. 11/390,682, and Internationalapplication No. PCT/US01/2881, are hereby incorporated by reference, intheir entirety for all purposes. While these publications providegeneral information about Id-1, it is understood that they do notpropose or describe the methods provided herein.

The cannabidiols and derivatives can be used alone or in combinationwith other cannabidiols or derivatives or with THC or derivativesthereof. Compositions and formulations of the cannabidiols, derivativesand combinations thereof or in combination with THC can be used to treatcancer and other cell proliferative disorders.

Non-psychoactive cannabinoids, compounds that do not interactefficiently with cannabinoid 1 (CB₁) and (CB₂) receptors, can modulatethe actions of Δ⁹-THC. The experiments described below, using multiplehuman (GBM) cells lines, compared the antiproliferative activity ofnon-psychoactive cannabinoids to synthetic and natural CB₁ and CB₂agonists. The activity of Δ⁹-THC was tested in combination with CBD. InU251 and SF126 cell lines, Δ⁹-THC in combination with a lowerconcentration of CBD, acted synergistically to inhibit GBM cell growthand induce apoptosis. The inhibitory properties of the combination werethe result of activation of CB₂ receptors and a corresponding increasein oxygen radical formation. The signal transduction mechanismsassociated with the effects of the combination treatment were differentfrom those observed with the individual compounds. The disclosuredemonstrates that the addition of CBD to Δ⁹-THC improves the overallpotency and efficacy of Δ⁹-THC in the treatment of patients with cellproliferative disorders such as, for example, GBM.

It was determined that Δ⁹-THC and CBD act synergistically to inhibit thegrowth of multiple GBM cell lines. It has been suggested thatnon-psychoactive cannabinoid constituents can either potentiate orinhibit the actions of Δ⁹-THC (Krantz et al., 1971; Jones and Pertwee,1972; Poddar et al., 1974; McPartland and Russo, 2001). The CB₁ and CB₂receptor agonist, Δ⁹-THC, can inhibit GBM growth in vitro and in vivo.CBD, a cannabinoid constituent with negligible affinity for CB₁ and CB₂receptors can also inhibit the growth of GBM in vitro and in vivo. Thedisclosure demonstrates that of the non-psychoactive cannabinoids, CBDwas the most potent inhibitor of GBM cell growth. Therefore, next thepositive and negative aspects of constituent interaction was determinedby testing multiple different concentration combinations of Δ⁹-THC andCBD in a 2×2 design. The three GBM cell lines that were originally usedto screen the antiproliferative activity of individual cannabinoid(Table 1) were next used to determine the effects of combinationtreatments. When applied in combination, Δ⁹-THC and CBD producessynergistic inhibition of cell growth in SF126 and U251 cells but not inU87 cells (FIG. 3, Panels A, B, C). Concentrations of Δ⁹-THC and CBDalone that produce only minimal effects on cell proliferation werecombined and further tested in a 2×2 factorial design in the positiveresponding cell lines (SF126 and U251) (FIG. 3, Panels D, E, F). Themost pronounced synergistic activity was observed with U251 cells,therefore, this cell lines was used to determine the mechanism of actionfor the combination effect. The 4:1 (1.7 μM: 0.4 μM) ratio of Δ⁹-THC andCBD was used for the remainder of the experiments.

The disclosure demonstrates that the combination treatment of Δ⁹-THC andCBD leads to the modulation of specific mitogen activated kinases(MAPK). The regulation of ERK, JNK, and p38 MAPK activity plays acritical role in controlling cell growth and apoptosis (Chang et al.,2003). Therefore in U251 cells, it was determined whether treatment witha combination of Δ⁹-THC and CBD could alter the activity of ERK, JNK,and p38 MAPK. Treatment with the combination of cannabinoids led to aprofound down-regulation of p-ERK but no significant change in total ERK(FIG. 4). Additionally, no inhibition of JNK or p38 MAPK activity wasobserved (FIG. 4). When U251 cells were treated with individualconcentration of Δ⁹-THC and CBD, instead of the combination, no changesin pERK were observed.

The disclosure further demonstrates that the combination treatment ofΔ⁹-THC and CBD induces apoptosis. Significant reductions in ERK activityhave been shown to lead to induction of apoptosis (Chang et al., 2003).The large reduction in GBM cell growth and ERK activity, observed in thepresence of the combination treatment of Δ⁹-THC and CBD, led to theprediction that there would be a corresponding modulation of programmedcell death. The measure of apoptosis (annexin staining) was used toprobe additional mechanism involved in the synergistic activity of theTHC/CBD combination. When Δ⁹-THC and CBD were combined a large increasein apoptosis was observed (FIG. 5). Separately 1.7 μM Δ⁹-THC and 0.4 μMCBD did not produce significant changes in apoptosis.

The disclosure demonstrates that the synergistic inhibitory effects ofcombination treatment are the result of CB₂ receptor activation andproduction of oxygen radicals. Depending on the cancer cell line andcompound used, studies have linked the inhibitory activity ofcannabinoids to activation of CB₁, CB₂, vanilloid (VR1) receptors, andthe production of oxygen radicals (Maccarrone et al., 2000; Jacobsson etal., 2001; Bifulco and Di Marzo, 2002; Guzman, 2003; Massi et al., 2006;McKallip et al., 2006).

Apoptosis produced by the combination of Δ⁹-THC and CBD was partiallyblocked by the CB₂ receptor antagonist, SR144528, and fully reversed inthe presences of α-tocopherol (oxygen radical scavenger) (FIG. 4). Thevanilloid receptor antagonist, capsazepine did not block any of theeffects observed with the combination treatment.

Accordingly, provided herein are compositions for treating cancer. Suchcompositions can comprise a combination of the cannabinoids cannabidiol(CBD) and tetrahydrocannabinol (THC).

In another embodiment, the composition can further include a compoundsuitable for treating a cell proliferation disorder, such as paclitaxel.

In another embodiment, a method of treating cancer in a subject,comprises administering to a subject in need of such treatment atherapeutically effective amount of a composition consisting essentiallyof a combination of the cannabinoids cannabidiol (CBD) andtetrahydrocannabinol (THC).

For administration to a subject, compositions are typically incorporatedinto pharmaceutical compositions suitable for administration.

Such compositions typically comprise compounds and a pharmaceuticallyacceptable carrier. Pharmaceutically acceptable carriers include any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

A pharmaceutical composition of the disclosure is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfate; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it is preferable to include isotonic agents, for example, sugars,polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

In one embodiment, compounds are prepared with carriers that willprotect the compound against rapid elimination from the body, such as acontrolled release formulation, including implants and microencapsulateddelivery systems. Biodegradable, biocompatible polymers can be used,such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid,collagen, polyorthoesters, and polylactic acid. Methods for preparationof such formulations should be apparent to those skilled in the art. Thematerials can also be obtained commercially from Alza Corporation andNova Pharmaceuticals, Inc. Liposomal suspensions (including liposomestargeted to infected cells with monoclonal antibodies to viral antigens)can also be used as pharmaceutically acceptable carriers. These can beprepared according to methods known to those skilled in the art, forexample, as described in U.S. Pat. No. 4,522,811.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose can beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma can bemeasured, for example, by high performance liquid chromatography.

The disclosure provides a method of using cannabinoids, or derivativesthereof (e.g., cannabidiols), as described herein, to treat cellproliferative disorders and metastasis. The cannabinoids and cannabidiolderivatives can be administered alone or in a pharmaceuticallyacceptable carrier. In other embodiments, the cannabinoids orcannabidiol derivatives can be administered in combination with a secondbiological active agent. In one embodiment, the second biological activeagent is a THC compound or derivative. In yet another embodiment, thesecond biological active agent is an anticancer drug. Suitableanticancer drugs can be selected from the group consisting ofmethotrexate (Abitrexate®), fluorouracil (Adrucil®), hydroxyurea(Hydrea®), mercaptopurine (Purinethol®), cisplatin (Platinol®),daunorubicin (Cerubidine®), doxorubicin (Adriamycin®), etoposide(VePesid®), Vinblastine (Velban®), Vincristine (Oncovin®) and Pacitaxel(Taxol®).

A “cell proliferative disorder” is any cellular disorder in which thecells proliferate more rapidly than normal tissue growth. Thus a“proliferating cell” is a cell that is proliferating more rapidly thannormal cells. The proliferative disorder, includes but is not limited toneoplasms. A “neoplasm” is an abnormal tissue growth, generally forminga distinct mass that grows by cellular proliferation more rapidly thannormal tissue growth. Neoplasms show partial or total lack of structuralorganization and functional coordination with normal tissue. These canbe broadly classified into three major types. Malignant neoplasmsarising from epithelial structures are called carcinomas, malignantneoplasms that originate from connective tissues such as muscle,cartilage, fat or bone are called sarcomas and malignant tumorsaffecting hematopoetic structures (structures pertaining to theformation of blood cells) including components of the immune system, arecalled leukemias and lymphomas. A tumor is the neoplastic growth of thedisease cancer. As used herein, a neoplasm, also referred to as a“tumor”, is intended to encompass hematopoietic neoplasms as well assolid neoplasms. Other proliferative disorders include, but are notlimited to neurofibromatosis, melanoma, breast cancers, head and neckcancers (e.g., brain cancers such as glioblastoma multiforme),gastrointestinal cancers and the like. A cancer generally refers to anyneoplastic disorder, including such cellular disorders as, for example,brain cancer, glioblasoma multiforme (GBM), renal cell cancer, Kaposi'ssarcoma, chronic leukemia, breast cancer, sarcoma, ovarian carcinoma,rectal cancer, throat cancer, melanoma, colon cancer, bladder cancer,mastocytoma, lung cancer and gastrointestinal or stomach cancer.

Metastasis is the final and often fatal step in the progression ofbreast cancer. Currently available therapeutic strategies at this stageof cancer progression are often non-specific, have only marginalefficacy and are highly toxic. This is in part due to the lack ofknowledge about the molecular mechanisms regulating the development ofaggressive cancers. Therapeutic approaches targeting only specificmechanisms involved in the development of aggressive breast cancers areurgently need. The expectation would be that this strategy would reduceunwanted toxicities associated with the therapy itself.

“Metastasis” generally refers to a multi-step process by whichaggressive cancer cells spread out of the primary tissue and into othertissues of the body. Aggressive cancer cells that are nourished throughangiogenesis, can migrate out of the primary tissue, and invade into theblood stream. These migratory aggressive cancer cells can remain vitalby escaping the immune response, and consequently evade the blood streamand invade other tissues of the body. These cells can then proliferateto create secondary tumors.

Human GBMs are highly heterogeneous and vary in their response totherapeutic treatments. This disclosure describes how this heterogeneityis reflected in the response of multiple aggressive GBM cancers celllines to the antiproliferative activity of synthetic and naturallyoccurring cannabinoids. Additionally, this disclosure discusses otherconstituents of marijuana that modulate the ability Δ⁹-THC to inhibitGBM cell growth and induce apoptosis. The disclosure demonstrates thatthe addition of CBD to Δ⁹-THC improves the overall potency and efficacyof Δ⁹-THC in the treatment of cancer.

In general, provided herein are methods for treating cancer byadministering to a subject in need of such treatment a therapeuticallyeffective amount of a composition consisting essentially of acombination of the cannabinoids cannabidiol (CBD) andtetrahydrocannabinol (THC). The methods include using a pharmaceuticalcomposition that includes a combination of the cannabinoids cannabidiol(CBD) and tetrahydrocannabinol (THC).

A subject generally refers to mammals such as human patients andnon-human primates, as well as experimental animals such as rabbits,rats, and mice, and other animals. Animals include all vertebrates,e.g., mammals and non-mammals, such as sheep, dogs, cows, chickens,amphibians, and reptiles.

“Treating” or “treatment” includes the administration of thecompositions, compounds or agents of the invention to prevent or delaythe onset of the symptoms, complications, or biochemical indicia of adisease, alleviating or ameliorating the symptoms or arresting orinhibiting further development of the disease, condition, or disorder(e.g., a disease or condition that is a result of immune systemover-activation). “Treating” further refers to any indicia of success inthe treatment or amelioration or prevention of the disease, condition,or disorder, including any objective or subjective parameter such asabatement; remission; diminishing of symptoms or making the diseasecondition more tolerable to the patient; slowing in the rate ofdegeneration or decline; or making the final point of degeneration lessdebilitating. The treatment or amelioration of symptoms can be based onobjective or subjective parameters; including the results of anexamination by a physician. Accordingly, the term “treating” includesthe administration of the compounds or agents of the disclosure toprevent or delay, to alleviate, or to arrest or inhibit development ofthe symptoms or conditions associated with cell proliferation, cancerand metastasis. The term “therapeutic effect” refers to the reduction,elimination, or prevention of the disease, symptoms of the disease, orside effects of the disease in the subject. “Treating” or “treatment”using the methods of the invention includes preventing the onset ofsymptoms in a subject that can be at increased risk of immune systemover-activation but does not yet experience or exhibit symptoms,inhibiting the symptoms of immune system over-activation (slowing orarresting its development), providing relief from the symptoms orside-effects of the condition, and relieving the symptoms of thecondition (causing regression). Treatment can be prophylactic (toprevent or delay the onset of the disease, or to prevent themanifestation of clinical or subclinical symptoms thereof) ortherapeutic suppression or alleviation of symptoms after themanifestation of the disease or condition.

The disclosure also provides methods for screening and developing alibrary of Id-1 inhibitors comprising derivatizing a compound of FormulaI, wherein the R group is modified. High throughput screeningmethodologies are particularly envisioned for the detection ofmodulators of expression of a target Id helix-loop-helix polypeptide,such as Id-1, described herein. Such high throughput screening methodstypically involve providing a combinatorial chemical or peptide librarycontaining a large number of potential therapeutic compounds (e.g.,modulator compounds). Such combinatorial chemical libraries or ligandlibraries are then screened in one or more assays to identify thoselibrary members (e.g., particular chemical species or subclasses) thatdisplay a desired characteristic activity. The compounds so identifiedcan serve as conventional lead compounds, or can themselves be used aspotential or actual therapeutics.

A combinatorial chemical library is a collection of diverse chemicalcompounds generated either by chemical synthesis or biologicalsynthesis, by combining a number of chemical building blocks (i.e.,reagents such as amino acids). As an example, a linear combinatoriallibrary, e.g., a polypeptide or peptide library, is formed by combininga set of chemical building blocks in every possible way for a givencompound length (i.e., the number of amino acids in a polypeptide orpeptide compound). Millions of chemical compounds can be synthesizedthrough such combinatorial mixing of chemical building blocks.

The preparation and screening of combinatorial chemical libraries iswell known to those having skill in the pertinent art. Combinatoriallibraries include, without limitation, peptide libraries (e.g. U.S. Pat.No. 5,010,175; Furka, 1991, Int. J. Pept. Prot. Res., 37:487-493; andHoughton et al., 1991, Nature, 354:84-88). Other chemistries forgenerating chemical diversity libraries can also be used. Nonlimitingexamples of chemical diversity library chemistries include, peptoids(PCT Publication No. WO 91/019735), encoded peptides (PCT PublicationNo. WO 93/20242), random bio-oligomers (PCT Publication No. WO92/00091), benzodiazepines (U.S. Pat. No. 5,288,514), diversomers suchas hydantoins, benzodiazepines and dipeptides (Hobbs et al., 1993, Proc.Natl. Acad. Sci. USA, 90:6909-6913), vinylogous polypeptides (Hagiharaet al., 1992, J. Amer. Chem. Soc., 114:6568), nonpeptidalpeptidomimetics with glucose scaffolding (Hirschmann et al., 1992, J.Amer. Chem. Soc., 114:9217-9218), analogous organic synthesis of smallcompound libraries (Chen et al., 1994, J. Amer. Chem. Soc., 116:2661),oligocarbamates (Cho et al., 1993, Science, 261:1303), and/or peptidylphosphonates (Campbell et al., 1994, J. Org. Chem., 59:658), nucleicacid libraries (see Ausubel, Berger and Sambrook, all supra), peptidenucleic acid libraries (U.S. Pat. No. 5,539,083), antibody libraries(e.g., Vaughn et al., 1996, Nature Biotechnology, 14(3):309-314) andPCT/US96/10287), carbohydrate libraries (e.g., Liang et al., 1996,Science, 274-1520-1522) and U.S. Pat. No. 5,593,853), small organicmolecule libraries (e.g., benzodiazepines, Baum C&EN, Jan. 18, 1993,page 33; and U.S. Pat. No. 5,288,514; isoprenoids, U.S. Pat. No.5,569,588; thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974;pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholinocompounds, U.S. Pat. No. 5,506,337; and the like).

Examples Cell Culture and Treatments

Human breast cancer cells lines MDA-MB231 and MDA-MB436 obtained fromthe ATCC were used. To prepare the MDA-MB231-Id-1 cells, cells wereinfected with a pLXSN-Id-1 sense expression vector. In all experiments,the different cell populations were first cultured in RPMI mediacontaining 10% fetal bovine serum (FBS). On the first day of treatmentthe media was replaced with vehicle control or drug in RPMI and 0.1%FBS. The media with the appropriate compounds were replaced every 24 h.Δ⁹-THC, CBN, CBD, CBG, and CP55,940 were obtained from NIH through theNational Institute of Drug Abuse. WIN55,212-2 was purchased fromSigma/RBI (St. Louis, Mo.).

MTT Assay:

To quantify cell proliferation the MTT assay was used (Chemicon,Temecula, Calif.). Cells were seeded in 96 well plates. Upon completionof the drug treatments, cells were incubated at 37° C. with MTT for 4 h,and then isopropanol with 0.04 N HCl was added and the absorbance wasread after 1 h in a plate reader with a test wavelength of 570 nm. Theabsorbance of the media alone at 570 nm was subtracted, and % controlwas calculated as the absorbance of the treated cells/control cells×100.

Boyden Chamber Invasion Assay:

assays were performed in modified Boyden Chambers (BD Biosciences, SanDiego, Calif.). Cells at 1.5×10⁴ per well were added to the upperchamber in 500 μl of serum-free medium supplemented with insulin (5ng/ml). The lower chamber was filled with 500 μl of conditioned mediumfrom fibroblasts. After a 20 h incubation, cells were fixed and stained.Cells that remained in the Matrigel or attached to the upper side of thefilter were removed with cotton tips. Invasive breast cancer cells onthe lower side of the filter were counted using a light microscope.

Quantitative Western Analysis:

Proteins were separated by SDS/PAGE, blotted on Immobilon membrane, andprobed with anti-Id-1 and the appropriate secondary antibody. Bandintensity values were obtained directly from the blot using AlphaeaseFCsoftware (San Leandro, Calif.) or from film using Image-J (NIH, MD). Asa normalization control for loading, blots were stripped and re-probedwith mouse alpha-tubulin (Abeam, Cambridge, Mass.).

Polymerase Chain Reaction:

Total cellular RNA was isolated from breast cancer cells treated withvehicle control or with CBD. Transcripts for Id-1 and for β-actin werereverse transcribed using SuperscriptII Reverse TranscriptaseII(Gibco-BRL), and polymerase chain reaction performed. The 5′ and 3′ PCRprimers were AGGTGGTGCGCTGTCTGTCT (SEQ ID NO:1) and TAATTCCTCTTGCCCCCTGG(SEQ ID NO:2) for Id-1; and GCGGGAAATCGTGCGTGACATT (SEQ ID NO:3) andGATGGAGTTGAAGGTAGTTTCGTG (SEQ ID NO:4) for β-actin. PCR was performed inbuffer containing 1 μM of each of the 5′ and 3′ PCR primer and 0.5 U ofTaq polymerase using 25 cycles for amplification of Id-1 and β-actincDNAs. The cycle conditions were 45 sec denaturation at 94° C., 45 secannealing at 55° C., and 1 min extension at 72° C.

Id-1 Promoter Reporter Assays:

A SacI-BspHI fragment of 2.2 kb corresponding to the 5′ upstream regionof human Id-1 gene and driving a luciferase gene in a PGL-3 vector(Promega) has already been described (Id-1-sbsluc). Cells were plated insix well dishes in medium supplemented with 10% FBS and 5 μg/ml insulin.After 24 h, cells were cotransfected with 6 μg of luciferase reporterplasmids and 2 μg of pCMVβ (Clontech) using superfect reagent (Qiagen).pCMVβ contained bacterial β-galactosidase and served to control forvariation in transfection efficiency. 3 h after transfection, the cellswere rinsed twice with PBS and were cultured in the absence or presenceof CBD for 48-72 h. Cell pellets were lysed in 80 μl of reporter lysisbuffer (Promega) for 10 min at room temperature. Lysed cells werecentrifuged and supernatants harvested. Luciferase and β-gal assays wereperformed using Luciferase Assay System (Promega), β-Gal Assay Kit(Clontech) and a 2010 luminometer (Pharmingen).

Statistical Analysis:

The IC₅₀ values with corresponding 95% confidence limits were comparedby analysis of logged data (GraphPad Prism, San Diego, Calif.). Whenjust the confidence limits of the IC₅₀ values overlapped significantdifferences were determined using unpaired Student's t-test. Significantdifferences were also determined (Prism) using ANOVA or the unpairedStudent's t-test, where suitable. Bonferroni-Dunn post-hoc analyses wereconducted when appropriate. P values <0.05 defined statisticalsignificance.

The ability of CBD to regulate the expression of key genes that controlbreast cancer cell aggressiveness and invasiveness was determined. Apotential candidate protein that could mediate the effects of CBD onboth phenotypes was the helix-loop-helix protein Id-1. It was determinedthat treatment of MDA-MB231 cells with CBD led to aconcentration-dependent inhibition of Id-1 protein expression (FIG. 1,panel B and panel C). The inhibitory effect of CBD on Id-1 expressionoccurred at concentrations as low as 100 nM. CBD was more effective atreducing Id-1 protein expression compared to other cannabinoid compounds(FIG. 1, panel C). The CBD concentrations effective at inhibiting Id-1expression correlated with those used to inhibit the proliferative andinvasive phenotype of MDA-MB231 cells. Furthermore, the time periodneeded to observe the down-regulation of Id-1 protein in the presence ofCBD correlated with the inhibitory effects of CBD on the aggressivenessand invasiveness of MDA-MB231 cells (FIG. 1, panel D).

The ability of CBD to modulate Id-1 gene expression was determinedReferring to FIG. 2, panel A, Id-1 mRNA expression was significantlyreduced upon treatment with CBD. To determine if this effect was due tothe inhibition of transcription, a construct was used that contained theId-1 promoter fused to a luciferase reporter in a PGL-3 basic vector.This construct was transiently transfected into MDA-MB231 cells.Twenty-four hours after transfection, MDA-MB231-Id-1-luc cells weretreated with CBD for 2 or 3 days and luciferase activity was measured(FIG. 2, panel B and panel C). Transfection efficiency and analysis ofequal amounts of total protein were controlled by contransfection of thecells with pCMVB containing β-galactosidase. Treatment with CBD resultedin a significant inhibition of luciferase activity. This effect wastime-dependent with the greatest inhibition occurring on day 3. Thesefindings correlated to the data obtained when the expression of the Id-1protein was assessed by Western analysis.

To determine if Id-1 represented a key mediator of CBD effects in highlyaggressive breast cancer cells, Id-1 was constitutively expressed intoMDA-MB231 cells (+Id1 as described in FIG. 7). The ectopic Id-1 gene,which is not under the control of the endogenous promoter, wasintroduced in the cells using the pLXSN retroviral vector. As a control,cells were infected with an empty pLXSN vector (−Id1). In control cells,treatment with CBD led to a significant reduction in cell invasiveness(FIG. 7, panel A (upper panels) and FIG. 7, panel B). Western blottingconfirmed the down-regulation of Id-1 expression in this control cellline (FIG. 7, panel C). In contrast to these results, CBD did notinhibit cell invasiveness (FIG. 7, panel A (lower panels) and FIG. 7,panel B) or Id-1 expression (FIG. 7, panel C) in MDA-MB231+Id1 cellsthat ectopically expressed Id-1.

Invasion is also an important step towards brain cancer progression. Thedisclosure also provide methods and compositions for the treatment ofbrain cancer progression. Therefore, the ability of CBD to reduce thegrowth and invasiveness activity of glioblastoma muliforme (GBM) cancercells was tested. Multiple glioblastoma muliforme (GBM) cell lines weretreated for three days and IC₅₀ values were calculated and provided inTable 2 below:

Cell Line CBD IC₅₀ SF126 1.2 (1.1-1.3) U87 0.7 (0.5-1.0) U251 0.6(0.5-0.7)

IC₅₀ values for the antiproliferative effects of CBD were calculated inmultiple GBM cell lines over a three day treatment. Cell proliferationwas assessed using the MTT assay. Data are the means and corresponding95% confidence limits of at least three experiments. IC₅₀ values arereported in μM.

It was determined that U251 cells were the most sensitive to theantiproliferative activity of CBD. CBD was also able to significantlyreduce the invasiveness of U251 cells (FIG. 10).

It was determined that treatment of U251 cells with CBD led to aconcentration-dependent inhibition of Id-1 protein expression (FIG. 11).

In order to determine the SAR between cannabinoids and the inhibition ofId-1, MDA-MB231 cells were treated for two days with multiplecannabinoid compounds and Id-1 protein levels were assessed. Thecompounds used included: 1) Agonists and antagonists to the putativeabnormal (Abn)-CBD receptor, Abn-CBD and 01602; 2) a syntheticcannabinoid analog that has high affinity for CB₁ and CB₂ receptors,CP55,940; 3) natural cannabis constituents that have appreciableaffinity for CB₁ and CB₂ receptors, Δ⁹-THC and CBN (Pertwee, 1997; Jaraiet al., 1999). The greatest inhibition of Id-1 was observed in thepresence of CBD. Also a small inhibition of Id-1 was observed in thepresence of CP55, 940. No inhibition of Id-1 was observed in thepresence of Δ⁹-THC, 0-1602 and Abn-CBD.

The data demonstrates that cannabidiol (CBD) is an effective inhibitorof Id-1. The inhibition of Id-1 does not appear to be related to theputative Abn-CBD receptor. It also appears that the openedtetrahydropyran ring in CBD is only partially responsible for itsactivity, since Abn-CBD and 0-1602 did not inhibit Id-1. One potentialkey structure is the classical cannabinoid aliphatic side chain: aregion crucial for the activity of numerous classical and syntheticcannabinoids. CP55,940 and CBN can partially inhibit Id-1. In comparisonto the classical cannabinoid structure (Δ⁹-THC), each compound containsthe side chain region, and the cyclohexyl ring, however, the pyrane ringis removed in CP55,940. CP55,940 has an opened tetrahydropyran ringsimilar to CBD. The data suggests that a general structural component ofCBD, responsible for Id-1 inhibition, is the combination of the openedtetrahydropyran ring and the classical cannabinoid aliphatic side chain.

Cell Culture and Treatments:

The human GBM cell lines used were SF126, U87 and U251. Cell lines weremaintained at 37° C. and 5% CO₂. In all experiments, the different cellpopulations were first cultured in RPMI media containing 10% fetalbovine serum (FBS). On the first day of treatment the media was replacedwith vehicle control or drug in RPMI and 0.1% FBS. The media with theappropriate compounds were replaced every 24 h. Δ⁹-THC, CBN, CBD, CBG,and CP55,940 were obtained from NIH through the National Institute ofDrug Abuse. WIN55,212-2 was purchased from Sigma/RBI (St. Louis, Mo.).

MTT Assay:

To quantify cell proliferation the3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrasodium bromide (MTT)assay was used (Chemicon, Temecula, Calif.). Cells were seeded in 96well plates at 1×10³ cells/well for seven day experiments and 3×10³cells per cm² for three day experiments to obtain optimal cell densitythroughout the experiment. Cells were incubated at 37° C. with MTT forfour hours, and then isopropanol with 0.04 N HCl was added and theabsorbance was read after one hour in a plate reader with a testwavelength of 570 nm. The absorbance of the media alone at 570 nm wassubtracted, and % control was calculated as the absorbance of thetreated cells/control cells×100.

Apoptosis:

Cells were grown in 6 well culture dishes and were treated with theappropriate compounds every 24 hours for three days. The cells weretrypsinized, washed with PBS, and processed for labeling withfluorescein-tagged UTP nucleotide and PI by use of an Apo-Directapoptosis kit obtained from Phoenix Flow Systems (San Diego, Calif.) andwas used according to the manufacturer's protocol. The labeled cellswere analyzed by flow cytometry. Cell Flow Cytometry in combination withPI and annexin staining was used to quantify the percentage of cellsundergoing apoptosis in control and treatment groups. % control wascalculated as annexin positive staining in treated cells/controlcells×100. PI staining was used to distinguish necrotic cells from thoseundergoing apoptosis.

Quantitative Western Analysis:

Cells cultured and treated in 6-well dishes were washed twice with coldPBS. Lysis buffer was added and cells lysed by freezing for 10 min at−70° C. and then thawing. Cell lysate was collected and theconcentration was determined using Bradford reagent. Equal amounts ofprotein were heated to 90° C. in Laemmli sample buffer withβ-mercaptoethanol, and loaded onto a precast SDS-PAGE gel (Bio-RadLaboratories, Hercules, Calif.). Protein was transferred to an Immobilonmembrane (Millipore, Billerica, Mass.) overnight at 2-4° C. Blots werethen blocked with 5% nonfat dry milk in TBS+Tween for 1 h. Primaryantibody from Millipore (rabbit anti-phospho-JNK, rabbitanti-phospho-p38, rabbit anti-phospho-ERK1/2 and rabbit anti-ERK1/2, 1mcg/mL) in blocking buffer was then added for 1 h. The blots were thenrinsed three times 10 min with TBS+Tween. Secondary antibody (DonkeyAnti-Rabbit IgG, Jackson Immunoresearch, West Grove, Pa.) was thenadded. Blots were incubated for 45 min and then washed 4 times for 15min each. The blots were developed with SuperSignal Femto (Pierce,Rockford, Ill.), and imaged on either a Fluorchem 8900 (Alpha Innotech,San Leandro, Calif.) or ECL Hyperfilm (Amersham-Pharmacia, Piscataway,N.J.). Band intensity values were obtained (after backgroundsubtraction) directly from the Fluorchem 800 using AlphaeaseFC software(San Leandro, Calif.) or from film using Image-J (NIH, MD). As anormalization control for loading, blots were stripped and re-probedwith mouse alpha-tubulin (Abcam, Cambridge, Mass.) and goat anti-mouseIgG (Jackson Immunoresearch) for the primary and secondary antibodies,respectively.

Cell Cycle Analysis.

U251 cells were grown in Petri dishes (100 mm×15 mm) and received drugtreatments for 2 days. On the third day, the cells were harvested andcentrifuged at 1200 rpm for 5 minutes. The pellet was washed 1× withPBS+1% BSA, and centrifuged again. The pellet was resuspended in 0.5 mlof 2% paraformaldehyde (diluted with PBS) and left to fix overnight atroom temperature. The next day the cells were pelleted and resuspendedin 0.5 ml 0.3% Triton in PBS and incubated for 5 minutes at roomtemperature. The cells were then washed 2 times with PBS+1% BSA. Thecells were finally suspended in PBS (0.1% BSA) with 10 ug/ml PropidiumIodide and 100 μg/ml RNAse. The cells were incubated for 30 minutes atroom temperature before being stored at 4 C. Cell cycle was measuredusing a FACS Calibur using Cell Quest Pro and Modfit software.

Radical Oxygen Species (ROS) Measurements.

The production of cellular radical oxygen species (ROS)/H₂O₂ wasmeasured using 2′-7′Dichlorodihydrofluorescein (DCFH-DA, Sigma Aldrich).DCFH-DA is deacylated intracelluarlly into a non-fluorescent product,which reacts with intracellular ROS to produce 2′-7′Dichlorofluorescein.2′-7′Dichlorofluorescein remains trapped inside the cell, and can bemeasured quantitatively. U251 cells were plated onto 6 well dishes andreceived drug treatments for three days. On the third day, 2 μM DCFH-DAwas added to the media (MEM with 0.1% FBS) and the cells were incubatedwith DCFH-DA overnight. The next day, the cells were trypsinized, washedwith PBS, and the fluorescent intensity was measured using a FACS andcell quest pro software.

Statistical analysis: Treatment groups were divided into 1) no treatment(control), 2) Δ⁹-THC alone, 3) CBD alone, 4) Δ⁹-THC and CBD combined.Positive and negative aspects of constituent interaction were determinedin this 2×2 design using 2-way analysis of variance as described by(Slinker, 1998). In the proliferation assays, IC₅₀ values withcorresponding 95% confidence limits were calculated using non-linearanalysis of logged data (GraphPad Prism, San Diego, Calif.). Significantdifferences were also determined using ANOVA where suitable.Bonferroni-Dunn post-hoc analyses were conducted when appropriate. Pvalues <0.05 defined statistical significance.

Two commonly used CB₁ and CB₂ receptors agonists were chosen to studythe effect of cannabinoid treatment on the growth of three humanglioblastoma multiforme (GBM) cell lines. Δ⁹-THC, a natural cannabisconstituent, and WIN 55,212-2, a synthetic cannabinoid analog, have highaffinity for CB₁ and CB₂ receptors. Human GBM cells were treated withmultiple concentrations of Δ⁹-THC and WIN 55,212-2. Cell proliferationwas measured using the MTT assay and corresponding IC₅₀ values werecalculated (Table 3). SF126 cells overall were most sensitive to theantiproliferative effects of Δ⁹-THC and WIN 55,212-2. Cannabidiol (CBD),a natural cannabis compound that does not have appreciable affinity forCB₁ and CB₂ receptors, was also tested in the GBM cell line, SF126. TheIC₅₀ value was 0.73 μM (0.64-0.82).

TABLE 3 Δ⁹-THC and WIN 55,212-2 inhibit cell proliferation in GBM celllines.    

SF126 0.9 (0.7-1.4) 0.84 (0.74-0.95) U87 1.6 (1.0-2.4) 0.77 (0.65-0.90)U251  1.1 (0.84-1.4) 1.1 (0.97-1.3) IC₅₀ values for theantiproliferative effects of Δ⁹-THC and WIN 55,212-2 were calculated inthree GBM cell lines over a seven day treatment. Cell proliferation wasassessed using the MTT assay. Data are the means and corresponding 95%confidence limits of at least three experiments. IC₅₀ values arereported in μM.

Three groups of cannabinoid compounds were chosen for a broader analysisof antiproliferative activity in the single GBM cell line, SF126 (Table4). 1) Natural cannabis constituents that have affinity for CB₁ and CB₂receptors, Δ⁹-THC and CBN. 2) Synthetic cannabinoid analogs that havehigh affinity for CB₁ and CB₂ receptors, WIN 55,212-2 and CP55,940. 3)Natural cannabis constituents that do not have appreciable affinity forCB₁ and CB₂ receptors, CBD and CBG.

TABLE 4 Multiple classes of cannabinoids inhibit SF126 cellproliferation. Compound SF126

Δ⁹-THC 2.5 (1.8-3.4)

CBN 1.2 (0.9-1.6)

WIN55, 212-2 1.3 (1.2-1.4)

CP 55, 940 3.3 (2.9-3.7)

CBD 1.2 (1.1-1.3)

CBG 1.6 (1.5-1.7) IC₅₀ values for the antiproliferative effects ofcannabinoid agonists on SF126 cell growth over a three day treatmentwere obtained. Cell proliferation was assessed using the MTT assay. Dataare the means and corresponding 95% confidence limits of at least threeexperiments. IC₅₀ values are reported in μM.

Treatment periods were shortened to three days during experiments withadditional agonists since significant effect were observed at this timepoint. SF126 cells were treated with a range of concentrations ofmultiple cannabinoid agonists, and the corresponding IC₅₀ values werecalculated. The rank order of potencies was: CBD=CBN=WIN55,212-2>CBG>Δ⁹-THC=CP55940. Again, CBD was one of the most potentcompounds tested.

Δ⁹-THC and CBD act synergistically to inhibit the growth of multiple GBMcell lines. The CB₁ and CB₂ receptor agonist, Δ⁹-THC, can inhibit GBMgrowth in vitro and in vivo and is currently being used in a pilotclinical trial (Velasco et al., 2007). CBD, a cannabinoid constituentwith negligible affinity for CB₁ and CB₂ receptors can also inhibit thegrowth of GBM in vitro and in vivo (Massi et al., 2004; Massi et al.,2008). The data provided herein determined that of severalnon-psychoactive cannabinoids tested, CBD was overall the most potentinhibitor of GBM cell growth. The positive and negative aspects ofconstituent interaction were tested by analyzing the activity ofdifferent concentration combinations of Δ⁹-THC and CBD in a 2×2 design(FIG. 3). When applied in combination, Δ⁹-THC and CBD producedsynergistic inhibition of cell growth in SF126 and U251 cells but not inU87 cells (FIG. 3 A, B, C). Concentrations of Δ⁹-THC and CBD alone thatproduce only minimal effects on cell proliferation were again combinedand further tested in a 2×2 factorial design in the positive respondingcell lines (SF126 and U251) (FIG. 3 D, E, F). The most pronouncedsynergistic activity was observed with U251 cells. Therefore, this cellline was used in the remainder of the experiments.

In addition to uncontrolled cell growth, a hallmark phenotype ofaggressive GBM tumor cells is their ability to migrate away for theprimary tumor of origin and invade into neighboring CNS tissue.Experiments were performed to determine whether the addition of CBD toΔ⁹-THC would improve the activity of the compound to inhibit migrationand invasion through a reconstituted basement membrane in a Boydenchamber. Δ⁹-THC effectively inhibited the invasiveness of U251 cells.Additionally, Δ⁹-THC was significantly more potent at inhibiting U251cell invasiveness in comparison to the inhibition of cell growth andinduction of apoptosis. The predicted IC₅₀ for the ability of Δ⁹-THC toinhibit U251 cell invasiveness was 85 nM (49-150). Whereas both THC andCBD were able to inhibit U251 cell invasiveness, the combined additionof the compounds did not result in activity suggesting a synergisticinteraction.

Since Δ⁹-THC and CBD acted synergistically to inhibit GBM growth but notinvasiveness, experiments were focused on the antiproliferative activityof the combination treatment. The highly active 4:1 (1.7 μM: 0.4 μM)ratio of Δ⁹-THC and CBD (FIGS. 3E and F) was used as the standardcombination treatment for the remainder of the experiments.

The combination treatment of Δ⁹-THC and CBD leads to the modulation ofspecific mitogen activated kinases (MAPK). The regulation of ERK, JNK,and p38 MAPK activity plays a role in controlling cell growth andapoptosis. U251 cells were used to determine whether modulation of ERK,JNK, and p38 MAPK activity occurred. Treatment with the combination ofcannabinoids led to a profound down-regulation of p-ERK but produced nosignificant change in total ERK (FIG. 4A). Additionally, no inhibitionof JNK or p38 MAPK activity was observed (FIG. 4B). When U251 cells weretreated with individual concentration of Δ⁹-THC and CBD, instead of thecombination, no changes in pERK were observed (FIG. 4C).

The combination treatment of Δ⁹-THC and CBD inhibits cell cycle. Thelarge reduction in GBM cell growth and ERK activity, observed in thepresence of the combination treatment of Δ⁹-THC and CBD, suggested therewould be a corresponding modulation of the cell cycle and programmedcell death. Therefore, U251 cells were treated with Δ⁹-THC and CBD aloneor with the combination of the two, and cell cycle was analyzed usingcell flow cytometery (FIG. 8). The combination of Δ⁹-THC and CBDproduces an increase in the population of cells in G1 phase and adecrease in cells in S phase. Additionally, there was an increase in thepopulation of cells in the G2/M phase. These changes in G1, S and G2/Mphase are hallmarks of cell cycle arrest. When administered separately,1.7 μM of Δ⁹-THC and 0.4 μM CBD both produced increases in thepopulation of cells in G1 and G2/M phase and decreases in cells in Sphase. Albeit, the magnitude of these effects was reduced compared tothose observed with the combination treatment.

Next apoptosis was measured using cell flow cytometery. When Δ⁹-THC andCBD were combined a large increase in apoptosis was observed (FIG. 5).Separately 1.7 μM Δ⁹-THC and 0.4 μM CBD did not produce significantchanges in apoptosis.

Apoptosis produced by the combination of Δ⁹-THC and CBD was partiallyblocked by the CB₂ receptor antagonist, SR144528, but complete reversalwas observed in the presence of the anti-oxidant, α-tocopherol (TCP)(FIG. 6A). The concentrations of the individual cannabinoids (Δ⁹-THC andCBD) were next increased in order to attempt to match levels ofapoptosis produced by the combination treatment. The purpose of theseexperiments was to determine whether the compounds alone recruitedsimilar pathways as compared to the combination of Δ⁹-THC and CBD. WhenU251 cells were treated with Δ⁹-THC alone, the induction of apoptosiswas completely blocked by α-tocopherol and partially blocked by the CB2antagonist, SR144528 (FIG. 6B). However, Δ⁹-THC alone could not producethe level of apoptosis observed with the combination treatment (FIG. 6Aand FIG. 6B). This finding was not simply an issue of the treatmentconcentration used since continuing to increase levels of Δ⁹-THC did notproduce a greater induction of apoptosis. When U251 cells were treatedwith CBD alone, the induction of apoptosis was completely blocked byα-tocopherol but no reversal was observed with SR144528 (FIG. 6C); thisresult would be expected since CBD does not interact efficiently witheither CB₁ or CB₂ receptors.

The ability of the higher concentrations of Δ⁹-THC and CBD alone toinhibit p-ERK were also studied and compared to the combinationtreatment (FIG. 6D). Again, the combination treatment produced aprofound down regulation of p-ERK. However, the higher concentration ofΔ⁹-THC alone had no effect on p-ERK activity. The higher concentrationof CBD produced a small inhibition of p-ERK. This suggests the pathway(s) activated by the Δ⁹-THC and CBD combination that leads to p-ERKdown-regulation, is unique to the combination treatment. As predicted byα-tocopherol blockade, the combination of Δ⁹-THC and CBD produced asignificant increase in the formation of ROS as assessed by DCDHF-DAoxidation (FIG. 6E).

The combination treatment of Δ⁹-THC and CBD produces the activation ofmultiple caspases. Caspases play a primary role in the regulation ofprogrammed cell death. Therefore, multiple caspase pathways wereevaluated to determine mechanisms by which the combination treatmentincreased apoptosis (FIG. 9). Treatment with the combination of Δ⁹-THCand CBD led to a significant up-regulation of caspase 3, 7, and 9activities as well as an increase in PARP. Small increases in theactivity of caspase 7, caspase 9 and PARP but not caspase 3 wereobserved when U251 cells were treated with the individual concentrationof Δ⁹-THC. In the presence of CBD alone no changes in caspase activitywere observed.

A wide range of cannabinoids inhibit the proliferation of human GBMcells. In addition to testing Δ⁹-THC, the analysis included thenon-psychoactive cannabis constituents CBD, CBN and CBG. Overall, CBDwas the most potent inhibitor tested.

Combining Δ⁹-THC and CBD together resulted in a synergistic increase inthe inhibition GBM growth and produced significant increases inapoptosis. This synergistic activity occurred in two of three GBM celllines tested. The synergistic inhibition of GBM cell growth was in partthe result of a greater amount of apoptosis being produced in presenceof the combination compared to administration of Δ⁹-THC alone. Treatmentof U251 cells with the combination of cannabinoids led to a profounddown-regulation of ERK activity, but not p38 MAPK and JNK1/2. Thereduction of ERK activity was specific for the combination treatmentindicating that all the effects observed were not simply due to anincrease in potency of Δ⁹-THC upon co-application with CBD. The specificreduction in ERK activity, observed in the presence of the combinationtreatment, may be one of the primary mechanisms leading to thesynergistic increase in inhibition of GBM cell growth and the inductionof apoptosis. Δ⁹-THC was also effective at inhibiting the invasivenessof U251 cells, however, there was no suggestion of a synergisticinteraction upon addition of CBD.

An increase in apoptosis produced by the combination of Δ⁹-THC/CBD waspartially dependent on CB₂ receptor activation. Apoptosis produce bytreatment of Δ⁹-THC alone was also partially dependent on CB₂ receptoractivation. Importantly, the induction of apoptosis in the presence ofthe combination treatment was significantly greater than that observedwith Δ⁹-THC alone. Apoptosis produced by CBD in U251 cells was notdependent CB₂ receptor activation. Comparable results with CBD were alsoobserved using another GBM cell line, SF126 (data not shown). Apoptosisproduced by the combination of Δ⁹-THC and CBD was greatly dependent onthe production of oxidative stress and resulted in the activation ofboth extrinsic and extrinsic caspase pathways.

Δ⁹-THC and CBD activate unique pathways in GBM cells that ultimatelyculminate in inhibition of cancer cell growth and invasion as well asinduction of cell death. The data presented here show that thesynergistic activity of the combination treatment is due in part to aspecific convergence of distinct pathways controlled by the individualcompounds. This convergence of inhibitory pathways unique to Δ⁹-THC andCBD leads to an overall synergistic reduction of GBM cell growth andsurvival. Combinations, compared to individual drug treatments withspecific cannabinoid-based compounds may represent a significantimprovement for the treatment of patients with GBM. These synergisticeffects may also be present in additional cancers. With the discovery ofa specific molecular mechanism potentially explaining the synergisticeffects, additional combination treatments may able to be refined inorder to further improve antitumor activity.

The examples set forth above are provided to give those of ordinaryskill in the art a complete disclosure and description of how to makeand use the embodiments of the apparatus, systems and methods of theinvention, and are not intended to limit the scope of what the inventorsregard as their invention. Modifications of the above-described modesfor carrying out the invention that are obvious to persons of skill inthe art are intended to be within the scope of the following claims.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1-15. (canceled)
 16. A method of treating glioblastoma multiforme in ahuman, consisting essentially of administering to said human in need ofsuch treatment a therapeutically effective amount of a combinationcomprising a cannabidiol (CBD) and a tetrahydrocannabinol (THC).
 17. Themethod of claim 16, wherein the CBD and THC are in a ratio of 1:1. 18.The method of claim 16, wherein the treatment of the glioblastomamultiforme in a human in need thereof is to reduce cell growth.
 19. Themethod of claim 16, wherein the THC and CBD are administered separatelyor in combination to said human.